GSM6932558: rna_mbd5_rep2; Danio rerio; RNA-Seq
For [Dataset 1], total RNAs were extracted with standard TRIzol protocol. For [Dataset 2], 76 hpf larval zebrafish were UV crosslinked and flash-frozen in liquid nitrogen. Pellets were thawed on ice and resuspended in 3 volume of ice-cold CLIP lysis buffer (50 mM HEPES pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, 1 × Halt™ Protease and Phosphatase Inhibitor Cocktail, 1 × RNaseOUT Recombinant Ribonuclease Inhibitor). Pellets were lysed by rotating at 4 °C for 15 minutes after passing through a 26 G needle (BD Biosciences). Embryo suspensions were sonicated on a bioruptor (Diagenode) with 30 s on/30 s off for 5 cycles. Lysates were cleared by centrifugation at 21,000 g for 15 minutes at 4 °C on a benchtop centrifuge. Supernatants were applied to Flag-antibody (Abcam) conjugated protein A beads (Invitrogen) and left overnight at 4 °C on an end-to-end rotor. Beads were washed extensively with 1 ml wash buffer (50 mM HEPES pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, 1 × Halt™ Protease and Phosphatase Inhibitor Cocktail, 1 × RNaseOUT Recombinant Ribonuclease Inhibitor) at 4 °C for 5 times. Protein-RNA complex conjugated to the beads were treated by 8 U/μL RNase T1 (Thermo Scientific) at 22 °C for 10 minutes with shaking. Input samples are digested in parallel. Then input and IP samples were separated on an SDS-PAGE gel and gel slices at corresponding size ranges were treated by proteinase K (Invitrogen) elution. RNA was recovered with TRIZol reagent (Invitrogen). Then T4 PNK (Thermo Scientific) end repair was performed with purified RNA before library constructions. For [Dataset 1], RNA libraries for input samples were constructed by SMARTer Stranded Total RNA-Seq Kit v2 (TaKaRa, 634417) with ribo-minus RNA as input.  For [Dataset 2], RNA libraries were constructed with NEB small RNA kit (NEB). Libraries were pooled and sequenced on a NovaSeq 6000 sequencer with paired-end mode 50 bp setting.